This application relates to improved oilseed Brassica plants and the seeds obtained from these plants, wherein such seeds have decreased fiber and increased oil and protein content. This invention also relates to methods for decreasing fiber content and increasing seed oil and protein levels in seeds from oilseed Brassica plants.
The term rapeseed is used to refer to a number of oilseed crop plant species within the genus brassica, including B. napus, B. rapa (syn. campestris), B. juncea and B. carinata. Spring and winter lines have evolved for B. napus and B. rapa, while only spring varieties of B. juncea are known. B. napus winter varieties are grown predominantly in northern Europe, China, and the northwest United States, whereas spring varieties predominate in Canada, northwest China, Denmark, and parts of Sweden. B. rapa has a shorter growing season than B. napus and this trait makes the spring varieties of this species suitable for the more severe climates of Sweden, Finland and Western Canada. B. juncea is grown extensively on the Indian subcontinent, while B. carinata is grown primarily in Ethiopia.
The taxonomic structure among members of the Brassica genus is complicated, as many of these species are able to cross pollinate naturally. Thus, there is a large amount of genetic variation available to oilseed Brassica breeders. The oilseed brassica species, B. napus, B. rapa, and B. juncea are closely related to one another, as well as to B. nigra, B. oleracea, and B. carinata. Cytological evidence indicates these species all originate from an extinct common ancestor which had 5 or 6 chromosomes, and that the high chromosome number Brassica species (B. napus, B. juncea, and B. carinata) originated as amphidiploid hybrids from combinations of low chromosome number species (B. nigra, B. rapa, and B. oleracea). The knowledge of the relationship between the various Brassica species creates possibilities for producing new synthetic oilseed Brassica material, as well as for transferring traits between the various related Brassica species.
Seedcoat color in rapeseed may be different depending on the particular species and variety of Brassica. Coat color is generally divided into two main classes, yellow or black (or dark brown), although varying shades of these colors, such as reddish brown and yellowish brown are also observed. Seeds with yellow coats have been found to have thinner hulls and thus less fiber and more oil and protein than varieties with dark color seed coats. Yellow-seeded rapeseed varieties are common in Asian countries, and in China, there is an abundance of yellow-seeded cultivars in production rapeseed, particularly in B. juncea and B. rapa varieties.
In order to improve the nutritional qualities of rapeseed oil, varieties have been developed which contain low erucic acid levels, as well as low glucosinolate levels. These varieties of B. napus and B. rapa have been termed xe2x80x9ccanolaxe2x80x9d by the Canadian breeders involved in their development, and the oils from these varieties are well accepted in the global vegetable oil markets. On the world markets, rapeseed oil does not derive from a particular species, and both high erucic acid and low erucic acid oils contribute to the edible oil supply. In 1996, about 14% of the global edible oil supply was from oilseed Brassica varieties.
The high protein content in its seed meal also makes rapeseed meal a valuable livestock feed, although the relatively high fiber content decreases its digestibility and decreases the value as an animal feed. Also, the presence of glucosinolates can decrease the value of the meal due to the deleterious effects of glucosinolates on growth and reproduction of livestock.
Improved oil and protein levels are primary objectives of rapeseed breeding programs. Thus, introduction of a yellow seed coat trait into canola varieties is desirable, in the interest of providing improvements in both the seed oil and protein levels. Integration of genes controlling seed pigmentation from related Brassica species into valuable oilseed Brassica varieties, such as canola varieties, is complicated by the fact that multiple recessive alleles are involved in the inheritance of yellow seed coats in presently available yellow coat lines.
Accordingly, there is a need in the art for yellow coat oilseed Brassica lines in which the trait for yellow seed coat can be easily transferred to other oilseed Brassica plants, and in particular to canola varieties. These lines can be used to accelerate the development of oilseed Brassica cultivars with improved oil, protein, and fiber contents.
Rapeseeds and oilseed Brassica plants producing rapeseeds, which have a yellow-seed coat are provided wherein the yellow-seed coat color is controlled by a single locus mutation. The rapeseed is characterized by having high levels of seed oil and protein and low levels of fiber and glucosinolate. The rape plants grown from the seed are also early maturing.
The plants and seed are useful for producing improved canola and rape varieties and for providing a source of valuable meal and oil products.
The invention is drawn to rapeseeds which have a yellow-seed coat, as well as to the plants producing such rapeseeds. The yellow-seed coat color is controlled by a single locus mutation. Thus, the trait can be easily transferred to desired lines without the complications associated with multiple gene inheritance.
Yellow seeds have a considerably thinner seed coat than black and brown ones. The thinner seed coat results in a reduced fiber content in the meal and an associated increase in seed oil and protein content as compared with normal levels of oil and protein. In seeds of the invention, seed oil levels are increased at least about 3%, preferably at least about 5%, more preferably at least about 10%.
Yellow-seeded genotypes generally have higher oil and protein concentrations in their seeds. The yellow-coated seeds of the invention display an increase in protein concentration. Protein levels are increased at least about 10%, preferably about 20%, more preferably about 30%.
Yellow-seeded genotypes typically show a decrease in fiber content. The yellow-coated seeds of the invention have decreased fiber content of at least about 2%, preferably about 5%, more preferably about 8%.
The seeds of the invention also exhibit a decrease in glucosinolate levels. Glucosinolate levels are decreased at least about 10%, preferably about 15%, more preferably about 20%. This reduction is important for utilizing the seed mean as animal feed.
Plants from the rapeseeds of the invention mature earlier than the parent lines from which these plants were developed. Earlier maturing varieties are particularly useful. For example, they find use where a short growing season is needed to avoid an environmental stress such as cold or drought or alternatively where multiple crops are grown. Various multiple cropping sequences are needed, particularly in China and India. In Western Canada, B. campestris varieties maturing in less than 100 days are required to escape frost damage. Additionally, short-duration rapeseed varieties are needed in Southern Australian to avoid late-season drought stress. By early maturing is intended that the plants mature at least about 2 days, preferably at least about 5 days, more preferably at least about a week or more early as compared to the parent lines from which the earlier maturing plant lines were developed.
Methods are available in the art for the production of plants and rapeseed of the invention. Generally, plant cells can be mutagenized and selected for those exhibiting the desired trait. Alternatively, the trait can be crossed into desired varieties using standard genetic methods known in the art.
For purposes of the invention, for mutagenesis and selection, plant cells are selected that are capable of regeneration such as seeds, microspores, ovules, pollen, vegetative parts, particularly microspores. For the most part, such plant cells can be selected from any variety of Brassicas, particularly those brassicas having desired agronomic traits.
Generally, methods are available in the art for the preparation of plant cells for mutagenesis. For collection of microspores for use in the invention, inflorescences are obtained from young plants and microspores collected. Microspore culture is an in vitro process whereby immature pollen grains (microspores) are mechanically isolated from anther tissue and are induced to develop directly into embryos rather than into mature pollen grains. The embryos originating from microspores have only one parental chromosome complement per cell. These embryos can be germinated and grown into plants. Generally, approximately 15-20% of the resulting plants are diploidized, i.e., are dihaploid-plants.
In oilseed brassicas, methods are known in the art for obtaining haploids through anther/microspore culture. See, for example, Plant Cell Culture in Crop Improvement edited by Giles and Sen, Plenum, New York (1982); Kameya and Hinata (1970) Jpn J Breed 20:82-87; Thomas and Wenzel (1975); Mathias, R (1988) Plant Breed 100:320-322; and George et al. (1987) 72-74, herein incorporated by reference. In Brassica, the developmental pathway for haploid induction normally involves the induction of microspore embryogenesis. See, Kott et al. (1988) Can J Bot 66:1665-1670. The use of microspore-derived haploids results in a substantial reduction in the time required to develop new varieties. Once the microspore or cell of interest has been obtained, methods are available in the art for mutagenesis. Such methods include radiation, chemical mutagenesis, or a combination of chemical and physical mutagenesis. Mutagenesis is carried out for a duration of time to accomplish genetic modifications, but not so long so as to destroy the viability of the cells and their ability to be regenerated into a plant.
Such modifications can be induced by physical mutagenesis such as radiation. Radiation mutagenesis includes mutagenesis carried out by use of physical means such as x-ray, gamma radiation, ultraviolet radiation, ionizing radiation, and the like. Gamma radiation may be supplied to the plant cells in a dosage of approximately 50 to 200 Krad., preferably from about 60 to about 90 Krad.
Chemical mutagenic agents include ethylmethylsulfonate (EMS), ethylnitrosourea (ENS), 2-aminopurine, 5-bromouracil (5-BU), alkylating agents, etc. Chemical mutagenesis involves the treatment of the cells with a dilute solution of the mutagen, typically about 0.01% to about 10% solution. For example, for the mutagenesis of microspores, a solution of about 0.01% to about 5%, preferably about 0.02% ethylmethylsulfonate is utilized. The microspores are treated with the chemical mutagen for one to several hours, typically about 2 to 3 hours.
The mutations may result in relatively small or even great changes in gene action. Ultimately, gene mutation is a physiochemical event that has the effect of increasing the number of different forms of the gene present in the population.
Haploid plants, mutagenized cells or microspores can be chemically treated to double the chromosome number. Such chemicals include colchicine which prevents the duplicated chromosomes from segregating by blocking spindle-fiber formation. While treatment time may vary, a time of one to several hours, preferably two to three hours is generally sufficient.
Development of homozygous lines by conventional methods involves repeated selfing and selection for several generations within a population. Early generation selection is limited by the heterozygosity of the material handled. Haploidization followed by chromosome doubling of early generation material can expedite the approach and shorten the breeding cycle. Thus, doubled haploid plants are useful for immediately obtaining homogenous plants.
Rape plants are regenerated from the treated cells using known techniques. See, McCormick et al. (1986) Plant Cell Reports, 5:81-84. The resulting rapeseeds may be planted in accordance with conventional growing procedures and following self-pollination rapeseed is formed.
Seed color character is generally scored on healthy plants at or near complete seed maturity. Since seed color in Brassica is determined by the genotype of maternal tissue, i.e., the testa, all F1 seeds of the Brassica plants of the present invention exhibit the same color as the female parent. Thus, when crossing the plants of the present invention to other varieties the successive F3 generation must be analyzed as the yellow coat color is recessive and the seed coat color is determined by the female parent. That is, the seed coat color in the progeny represents the genetic make up of the parent plants. Typically for a single locus trait, the F3 generation must be analyzed to select for those hybrids inheriting the yellow-seed coat trait. In a cross, F1 seed would be all black or yellow depending on whether the yellow seed coat variety was the male or female parent. F2 seed would be all black reflecting the heterozygous nature of the F1 plants. A segregation ratio of 3:1 in the F3 generation (black:yellow) is indicative of a single locus trait.
Care is taken in the growth and maintenance of the treated rapeseed. Particularly, pollination is carefully controlled and monitored. The resulting rapeseed is harvested and subjected to analysis, for seed coat color, fatty acid composition, protein composition, and the like.
Once a plant has been selected exhibiting the yellow coat color, the plant and seed can additionally be tested for other desirable characteristics. Thus, the seed oil can be analyzed for altered fatty acid content, rate of maturity, protein and fiber levels, and the like. For determination of the fatty acid composition of a given rapeseed, methods are available in the art for removal of the oil from the rapeseed and determination of the composition of the oil. Generally, the oil is removed from the rapeseeds by crushing the seeds and extraction of the oil. Nuclear magnetic resonance (NMR), gas liquid chromatography using a capillary column, and near infrared (NIR) spectroscopy can be used to measure the oil content. Generally, the oil is extracted as fatty acid methyl esters following reaction with methanol and sodium hydroxide. The resulting ester can be analyzed for fatty acid content. See, Daun et al. (1983), J. Amer. Oil. Chem. Soc. 60:1751-1754; Oil Crops of the World, edited by Robbelen et al., Toronto: McGraw-Hill (1989); and Starr et al. (1985) J Agric Sci Camb 104:317-323.
Brassica napus and Brassica campestris are dicotyledonous plants. Thus, the analysis for fatty acid composition may be carried out on a single outer cotyledon and the remaining halfseed can be retained for possible future germination. Methods are available in the art for the separation of the rapeseeds and the two halfseed.
After analysis of the fatty acid composition and seed coat color, seed are selected for further self pollination to achieve substantial genetic homogeneity. Such plants can then be used as breeding or other source material for the production of improved rape varieties.
Improved oil and protein concentrations are primary objectives in rapeseed breeding. Oil and protein levels can be further improved by crossing the plants of the invention with lines which have high oil and protein levels. Likewise, other characteristics may be improved by careful consideration of the parent plant.
Prior to the present application, light seed color has been associated with recessive genes at multiple loci. However, the present invention indicates that a yellow-seed coat rapeseed can be produced wherein the yellow-seed coat is controlled at a single locus.
The discovery of a single locus which is capable of conferring yellow seed color is beneficial for crossing such trait into other rape or canola lines, including high erucic acid lines, canola lines, and other elite lines or rape. The trait can be readily transferred into other plants within the same species by conventional plant breeding techniques including cross-pollination and selection of the progeny. Also, the desired traits can be transferred between species using the same convention plant breeding techniques involving pollen transfer and selection. See, for example, Brassica crops and wild allies biology and breeding, edited by S. Tsunada et al., Japan Scientific Press, Tokyo (1980); Physiological Potentials for Yield Improvement of Annual Oil and Protein Crops, edited by Diepenbrock and Becker, Blackwell Wissenschafts-Verlag Berlin, Vienna (1995); Canola and Rapeseed, edited by F. Shahidi, Van Nostrand Reinhold, New York (1990); and Breeding Oilseed Brassicas, edited by Labana et al., Narosa Publishing House, New Dehli (1993), herein incorporated by reference.
The yellow seed coat color trait can be readily transferred into other plants within the same Brassica napus or Brassica campestris species by conventional plant breeding techniques. Such techniques include cross-pollination and selection of the progeny. Such techniques can likewise be used to transfer the trait between the napus and campestris species. See, for example, Brassica Crops and Wild Allies Biology and Breeding, edited by S. Tsunada et al. Japan Scientific Press, Tokyo (1980). Commercially available campestris varieties include Tobin, Horizon, Colt, etc. Following the interspecific cross, members of the F1 generation are self-pollinated to produce F2 seed. Backcrossing is then conducted to obtain a euploid (n=10) campestris line exhibiting the desired trait. Additionally, protoplast fusion and nuclear transplant methods can be used to transfer the trait from one species to another. See, generally, xe2x80x9cFusion of Higher Plant Protoplastsxe2x80x9d by Albert W. Ruesink, Methods in Enzymology, Vol. LVIII, Jakoby and Pastan (eds), Academic Press, Inc., New York, N.Y. (1979), and the references cited therein; and Carlson et al. (1972), Proc. Natl. Acad. Sci. USA 69:2292.
In accordance with the present invention, a substantially uniform assemblage of rapeseed can be produced. Such seed can be used to produce a substantially uniform field of rape plants.
The relatively high content and quality of rapeseed protein make the seed a valuable raw material, not only for the oil industry but also for the feed industry. The factor most limiting utilization of rapeseed meal is the presence of gluscosinolates or the products formed by their cleavage. These compounds are responsible for the enlargement of thyroid glands and hemorrhagic liver syndrome in animals. These affects have limited the use of rapeseed meal, prompting efforts to breed new rapeseed varieties low in gluscosinolates.
Generally, the high fiber content of rapeseed meal contributes to the overall reduction of the feed value of the rapeseed meal. Furthermore, when edible protein products are made from rapeseed meal the dark color of black seed is a considerable problem. The black-seed coat gives an unpleasant grey color to protein products made from rapeseed meal. Therefore the reduction in seed coat color of the rapeseed of the invention is of interest from the point of view of protein quality as well as lecithin quality.
Methods are available for determination of the protein content of the seed. Traditionally, protein content has been determined by the Kjeldahl or micro Kjeldahl approach of acid digestion followed by steam distillation of the liberated nitrogen as ammonia, and back titration. Automated approaches are available using Kjel Foss or the Technicon autoanalyzer. NIR spectroscopy may also be utilized. See, for example, Gehrke et al. (1968) Technicon Symposium, Automation in Analytical Chemistry 1:239-251; Williams (1975) Serial Chem. 52:561-576; Thachuk, R. (1981) J. Am. Oil Chem. Soc. 58:819-822; Starr et al. (1985) J. Agric. Sci. Camb. 104:317-323; and Canola and Rapeseed, edited F. Shahidi, Van Nostrand Reinhold, N.Y., 1990, and the references cited therein.
In a particular embodiment of the invention, an improved rapeseed designated LFDF is disclosed. The rapeseed has yellow-seed coat wherein the seed coat color is controlled by a single locus. The locus has been designated LFDF. Seed of Brassica napus xe2x80x98212/86xe2x80x99 mutagenized LFDF, also known as E25-59 line, has been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 USA, now located at 10801 University Boulevard, Manassas, Va. 20110 USA, and given accession number 97875. The deposit was made on Feb. 11, 1997. The rapeseed exhibits high levels of seed oil and protein and low levels of glucosinolate and fiber. Oil extracted from seeds can be analyzed for lines that contain an oil of interest. Plants from the seed mature early. Generally, the plants mature at least 2 days earlier than the cultivars from which they are derived.
The seed and plants therefrom are useful for transferring the yellow-seed coat color into other rape lines, including high erucic acid lines, canola lines, and other elite lines. The plants are useful for the development of cultivars with improved oil, protein, and fiber concentrations.